The ADP/ATP carrier/Adenine nucleotide translocase (yeast AAC = mammalian ANT) mediates the 1:1 exchange of ADP into and ATP out of the mitochondrial matrix and is thus required for oxidative phosphorylation (OXPHOS). Recently, we demonstrated that the major ADP/ATP carrier in the yeast Saccharomyces cerevisiae, Aac2p, physically associates with the respiratory supercomplex but only in the context of mitochondrial membranes that contain the unique phospholipid cardiolipin (CL). ANT1 deficiencies and mutations have been linked to numerous diseases including hypertrophic cardiomyopathy. Moreover, there are a multitude of pathologies caused by alterations in CL metabolism including both inherited and diabetic cardiomyopathies. Given the critical importance of CL for the Aac2p interactome, we hypothesize that the CL-dependent ADP/ATP carrier interactome represents the mitochondrion's Achilles' heel in the multiple disease states that result from altered CL metabolism. The goal of the first specific aim is to identify mammalian ANT binding partners. Once an inventory of interacting proteins is determined, the functional importance of each interaction will be probed using stable cell lines expressing a transport-null disease allele of ANT1; and both a murine model and cell lines of the CL-based cardiomyopathy, Barth syndrome. Results from this aim will provide novel insight into the physiological importance of this critical component of OXPHOS for diverse mitochondrial functions. The second specific aim will determine the role of CL in establishing the interactome of the extended ADP/ATP carrier family. Specifically, the complex assembly of endogenous ANT2 will be ascertained in two CL-deficient mammalian cell models. In addition, the complex assembly of all four human ANT isoforms and two other yeast AAC isoforms will be determined in CL-null yeast. Ultimately, the high sequence homology between AAC isoforms will facilitate efforts to determine if Aac2p contains specific CL binding motifs and if so, define the minimal amino acid requirements. This information will in turn be used to define the relative importance of Aac2p-CL to overall OXPHOS efficiency. Finally, the third aim will biochemically interrogate the interaction between Aac2p and the respiratory supercomplex with the ultimate goal of defining the motifs within Aac2p responsible for this association. In addition, how this association promotes optimal OXPHOS will be dissected focusing on both sides of the interaction using transport-active, interaction-null and transport-null, interaction-active Aac2p variants. By defining the contributio of Aac2p- respiratory supercomplex to optimal mitochondrial function, results from this aim will provide keen insight into multiple OXPHOS disorders. Results from this application will significantly impact our understanding of the consequences of alterations in the ANT interactome that may occur due to mutations in ANT and/or perturbations in CL metabolism. In turn, a greater understanding of basic mechanisms contributing to cardiovascular disease, the number one cause of death in the United States, will be obtained.